The well known complications of blood transfusion namely incompatibility reactions, disease transmission, immunosuppression and the storage limitations of erythrocytes points to the need for the development of blood substitutes devoid of these shortcomings. Blood substitutes will have numerous applications provided they are safe, meet the viscosity and flow requirements, have long in vivo and shelf life and are cost effective. The products currently under development include perfluorocarbons and Hb-based oxygen carriers. Each of these preparations offer advantages and disadvantages but none appears to be useful for making an artificial blood substitute.
The present invention provides a modified hemoglobin and describes the use of the modified hemoglobin as a component of a blood substitute composition. It is well known that the hemoglobin molecule is present in erythrocytes and acts as the agent for the transport of oxygen in mammalian circulatory systems by binding and releasing oxygen. Hemoglobin is a conjugated protein with an approximate molecular weight of 64,000. It contains basic proteins, the globins and ferroprotoporphyrin or heme. It is essentially a tetramer consisting of two alpha chains each containing 141 amino acids and two beta chains each containing 146 amino acids. The binding site for oxygen in each of the monomers which make up the tetramer is the Fe.sup.+2 molecule in the heme molecule. The oxygen binding capability is modified by the presence of 2,3-di-phospho glycerate (2,3-DPG).The 2,3-DPG is reversibly attached to the central cavity of the Hb which is formed by the steric configuration of the hemoglobin molecule. It is known that when hemoglobin is separated from erythrocytes by hemolysis, it retains its ability to bind oxygen but loses its ability to readily release oxygen which is facilitated by the presence of 2,3-DPG. Even though free hemoglobin is commercially available as a genetically engineered material, its use as an oxygen carrier has not been possible because of its instability and the problem of the releasability of bound oxygen. In the blood circulation, free hemoglobin breaks down into its dimer and monomeric subunits which cannot be retained because of their relatively small size. These small fragments of hemoglobin are readily filtered by the kidneys and may pass through the subendotheilium. The fragments will also bind NO (endotheilial cell derived smooth muscle relaxing factor). The binding of NO causes elevation of the systemic and pulmonary vascular resistance.
The present invention provides a chemically modified form of hemoglobin that is stabilized and can efficiently bind and release oxygen. In addition the chemically modified hemoglobin may be polymerized to increase its molecular weight and increase its stability so that it will have a longer half life in the circulatory system and may be used as a stable oxygen transport mediator which is useful as the basis of a blood substitute. The chemical modification is achieved by the use of clofibric acid derivatives.